Age Related Change in Mitochondrial Angiotensin System and Mitochondrial Decline

项目摘要 衰老和线粒体功能下降密切相关。线粒体功能障碍的主要症状包括 活性氧的产生增加，每消耗O2产生的ATP分子减少， 凋亡增加。ATP的减少转化为细胞维持过程的能量降低 包括有丝分裂和周转。我们最近报道了一个功能性线粒体的鉴定， 血管紧张素系统（MAS），并发现衰老影响关键血管紧张素1型和2型的比例 线粒体受体（AT 1 R和AT 2 R），AT 1 R阻断剂可能部分逆转这些变化 改善线粒体功能重要的是，AT 1 R-/-小鼠的寿命延长了25%，部分原因是 通过线粒体数量的增加。衰老对线粒体基因（mt）表达的影响 AT 1 R和mtAT 2 R及其对线粒体功能障碍中年龄相关变化的作用尚未被证实。 以前研究过。新的初步证据表明，在老年小鼠中阻断AT 1 R可能会恢复年龄- 线粒体能量产生的相关下降，并通过改变p53- 诱导蛋白（MIEAP）表达。我们假设年龄相关的mtAT 1 R/AT 2 R比值增加， 通过增加活性氧（ROS）介导线粒体能量代谢下降 产生和受损的线粒体的受损消除。为了验证这一假设，我们提出了一个 对照组和氯沙坦（AT 1 R阻断剂）治疗组年轻人和老年人mtAT 1 R和mtAT 2 R的综合研究 （38月龄）C57 BL/6、AT 1-/-和AT 2-/-小鼠。将使用与年龄相关的心肌生物能量学衰竭作为 模型研究mtAT 1 R和mtAT 2 R的变化的影响，提出了具体的目标：1。以识别 外周血管紧张素系统（PAS）和MAS的年龄相关变化及其对改变 活性氮/氧物质ROS/RNS产生，使用Q-PCR、蛋白质印迹、共聚焦和电子显微镜， 显微镜ROS/RNS的产生将在来自所有受试者的分离的心肌细胞和线粒体中定量。 我们的动物群体使用特定的荧光探针。2.说明年龄相关变化的贡献 在PAS和MAS中，通过磁共振波谱和成像， 在基线和安慰剂或氯沙坦治疗四周后的活动物的心脏组织。MAS 将在来自所有动物的分离的心脏线粒体中研究对生物能量学变化的贡献 组3.研究PAS和MAS在线粒体生物发生、修复和凋亡中的作用， 通过使用装有P53活性报告物的心脏细胞系（H9 C2）测量细胞的清除， 过表达AT 1 R和/或AT 2 R对MIEAP、氧化应激、mtDNA损伤和线粒体自噬的影响。 还将比较对照组和LOS治疗组、年轻组和老年组（38个月）的相似结局 C57 BL/6、AT 1-/-和AT 2-/-小鼠。

Project Abstract Aging and decline in mitochondrial function are closely linked. Key signs of mitochondrial dysfunction include increased generation of reactive oxygen species, fewer ATP molecules produced per O2 consumed, and increased apoptosis. The reduction in ATP translates to lower energy for cellular maintenance processes including mitobiogenesis and turnover. We recently reported the identification of a functional Mitochondrial Angiotensin System (MAS), and found that aging impacts the ratio of key angiotensin type 1 and type 2 receptors in mitochondria (AT1R and AT2R), that AT1R blocking agents may partially reverse these changes and improve mitochondrial function. Importantly, AT1R-/- mice have an enhanced life span by 25%, in part through an increase in mitochondrial numbers. The effects of aging on the expression of mitochondrial (mt) AT1R and mtAT2R and their contribution to age-related changes in mitochondrial dysfunction has not been previously studied. New preliminary evidence suggests that AT1R blockade in old mice may restore the age- related decline in mitochondrial energy production and improve mitophagy efficiency via alterations in p53- inducible protein (MIEAP) expression. We hypothesize that an age-related increase in mtAT1R/AT2R ratio mediates declines in mitochondrial energy metabolism via increased reactive oxygen species (ROS) production and impaired elimination of damaged mitochondria. In order to test this hypothesis we propose a comprehensive study of mtAT1R and mtAT2R, in control and losartan (AT1R blocker) treated, young and aged (38-month old) C57BL/6, AT1-/- and AT2-/- mice. Will use age related cardiac muscle bioenergetics failure as a model to study the impact of changes in mtAT1R and mtAT2R following proposed specific aims: 1. To identify age-related changes in peripheral angiotensin system (PAS) and MAS and their contribution to altered reactive nitrogen/oxygen species ROS/RNS generation, using Q-PCR, western blot, confocal and electron microscopy. Generation of ROS/RNS will be quantified in isolated cardiomyocytes and mitochondria from all our animal groups using specific fluorescent probes. 2. To specify the contribution of age-related changes in PAS and MAS to bioenergetic dysfunction by magnetic resonance spectroscopy and imaging in the cardiac tissue of living animals at baseline and after four weeks of placebo or losartan treatment. MAS contributions to the changes in bioenergetics will studied in isolated cardiac mitochondria from all animal groups. 3. To investigate the role of the PAS and MAS on mitochondrial biogenesis, repair, and elimination by utilizing a cardiac cell line (H9C2) instrumented with a P53 activity reporter to measure the effects of over-expressing AT1R and/or AT2R on MIEAP, oxidative stress, mtDNA damage and mitophagy. Similar outcomes will also be compared between control and LOS treated, young and aged (38-month old) C57BL/6, AT1-/- and AT2-/- mice.